In vivo turnover and biodistribution of soluble AXL: implications for biomarker development

Soluble biomarkers are paramount to personalized medicine. However, the in vivo turnover and biodistribution of soluble proteins is seldom characterized. The cleaved extracellular domain of the AXL receptor (sAXL) is a prognostic biomarker in several diseases and a predictive marker of AXL targeting agents. Plasma sAXL reflects a balance between production in tissues with lymphatic transport into the circulation and removal from blood by degradation or excretion. It is unclear how this transport cycle affects plasma sAXL levels that are the metric for biomarker development. Radiolabeled mouse sAxl was monitored after intravenous injection to measure degradation and urinary excretion of sAxl, and after intradermal injection to mimic tissue or tumor production. sAxl was rapidly taken-up and degraded by the liver and kidney cortex. Surprisingly, intact sAxl was detectable in urine, indicating passage through the glomerular filter and a unique sampling opportunity. The structure of sAxl showed an elongated, flexible molecule with a length of 18 nm and a thickness of only 3 nm, allowing passage through the glomerulus and excretion into the urine. Intradermally injected sAxl passed through local and distant lymph nodes, followed by uptake in liver and kidney cortex. Low levels of sAxl were seen in the plasma, consistent with an extended transit time from local tissue to circulation. The rapid plasma clearance of sAxl suggests that steady-state levels in blood will sensitively and dynamically reflect the rate of production of sAxl in the tissues but will be influenced by perturbations of liver and kidney function.


Methods
Two different techniques were used for radioactive labelling of sAxl as explained in detail below.For labelling with 18 F, [ 18 F]Py-TFP acts as a prosthetic group that facilitates the attachment of the radioactive fluorine-18 to the protein through a lysine residue modification 1 .This method is known for its precision and ability to maintain the biological activity of the labeled protein.In the following text and in the main manuscript, 18 F[PyTFP]-sAxl is referred to as 18 F-sAxl, and the free tracer, whether it is bound to its prosthetic group or not, is denoted as 18 F.
The labeling of sAXL with [ 125 I] was accomplished using Iodogen reagent (1,3,4,6-tetrachloro-3α,6α-diphenylglycouril), which acts as an oxidative agent that facilitates the electrophilic substitution of iodine-125 onto the tyrosine residues of the protein.This method does not involve a prosthetic group in the conventional sense but instead uses a chemical reaction to directly label the protein.The Iodogen method is an indirect labeling technique where the Iodogen reagent remains separate from the protein, thus reducing potential oxidative damage.

Labelling sAxl with 18 F
When labeling mouse sAxl, two vials of lyophilized powder (100 μg each) were reconstituted to a concentration of 0.25 mg/ml by adding 0.4 ml of milli-Q water to each vial, in accordance with the manufacturer's recommendations.The s-Axl solution from both vials were then pooled into an Amicon® Ultra 4 mL Filter device (10 kDa molecular weight cutoff) along with 3.2 ml of 0.05 M Na2HPO4 in milli-Q water, adjusted to pH 8.5.The mixture was centrifuged to a final volume of 200 µl to remove potential contaminants and to ensure that the pH was suitable for 18 F labeling. 18F was of pharmaceutical grade and obtained from the cyclotron at Haukeland University Hospital, Bergen, Norway on the day of the labelling.Approximately 0.2 mg sAxl was labeled with 18 F for PET-imaging by using a similar approach as described by Olberg et al 1 .The purification and integrity assessment of 18 F-sAxl were conducted using a PD MidiTrap G-10 gravity column and size-exclusion chromatography HPLC, respectively, with specific columns and conditions as detailed previously 2 .The elution pattern of 18 F-sAxl, monitored by in line gamma detection, closely matched that of unlabeled sAxl.

Labelling sAxl with 125 I
125 I (74 MBq) was obtained from Perkin Elmer (Boston, USA).Batch/ Serial number was I0I2021H/1 as of date 01/20/2021 and was delivered at 74 MBq in a carrier-free, high pH (pH 12-14) formulation of Iodine-125 recommended for Iodogen labeling.A stock solution of sAxl in 200µl Tris Iodination Buffer (25 mM Tris-HCl, pH 7.5, 0.4 M NaCl) was prepared using 200 µg of lyophilized sAxl following a similar procedure as described for 18 F labeling.Then, 60 µl of Tris Iodination Buffer and 40 µl of the sAxl stock (approximately 0.8 nmol sAxl) were transferred to the bottom of a 1.8 ml Nunc Cryotube at room temperature.One Pierce™ Pre-Coated Iodination Tube was washed with 1 ml Tris Iodination Buffer, decanted and 100 μl buffer together with 10 μl (37 MBq) carrier free Na 125 I (643 GBq/mg) were added to the bottom of the tube.
125 I-ions were allowed to activate to 125 I+ ions by swirling the iodination tube every 30 s for 6 min at room temperature.Activated 125 I was immediately transferred into the tube containing sAxl for incorporation into the tyrosine residues by electrophilic attack of its ortho ring position.The tube was flicked every 30 s for 8 min and the reaction stopped by adding 50 µl scavenging buffer (10 mg tyrosine/ml in Tris Iodination Buffer, pH 7.4).The solution was mixed and incubated for 5 minutes, with flicking at 1 and 4 minutes, before being transferred to an Amicon® Ultra 15 mL Filter device (10 kDa cutoff).It was then diluted to a total volume of 15 ml with Tris/NaCl/EDTA Buffer (25 mM Tris-HCl, pH 7.5, 0.4 M NaCl, 5 mM EDTA, 0.05% sodium azide) and stored at 4 °C within the device.Shortly before use, the 125 I-sAxl solution was concentrated to 200 µl through centrifugation and subsequently washed twice with 14.8 ml of 0.9% saline to remove any unincorporated 125 I. Low molecular weight radioactivity accounted for <0.3% of the total activity.The radioactivity was determined in a Gammacounter: Perkin Elmer Wallac Wizzard 1470 auto gamma counter (USA).

PET analysis of 18 F-sAxl injections
Dynamic PET scan was performed at the Molecular Imaging Center at University of Bergen on a nanoScan PET/ CT from Mediso (Mediso Ltd, Budapest, Hungary) during isoflurane anesthesia.In vivo experiments were performed immediately following the labelling process and purification.The integrity of the tracer was then confirmed by size-exclusion chromatography HPLC 2 .
PET scan acquisition parameters were as follows: Field of view 9.6 cm in the axial direction and 10 cm in the transaxial direction, 1:5 coincidence mode and normal count rate mode 2 .The body temperature was maintained at 37 °C throughout the whole procedure.Reconstruction of the PET data was performed with 3D OSEM and 1:5 coincidence mode, no filtering, attenuation correction (from a helical CT-scan, 50 kVp, 300 ms, 360 projections reconstructed using Ram-Lak filter), decay correction and normalization of detectors.All images were exported to Image J software (v.1.47 h, Fiji distribution) 3 for further analysis.
PET experiments were analyzed using InterView Fusion version 3.0.1 (Mediso, Budapest, Hungary), with Volume of Interest (VOI) tools for different organs detailed below and in Supplemental Fig. 1.
Blood: Radioactivity was sampled over the large abdominal blood vessels, fitting a VOI-sphere in the center of the lumen across three planes (sagittal, axial and coronal).
Lymph node: 18 F-sAxl concentration in lymph nodes was measured using two methods: 1) By calculating the Area Under the Curve (AUC) for mean activity measured as Standardized Uptake Value (SUV) along two orthogonal transects that intersect at the node's peak activity, and 2) By determining the mean activity (SUV) of the lymph node using an isocount 3D Region of Interest tool.The average 18 F-sAxl activity (SUV) was then calculated as the mean of these two results.The total radioactivity (in Bq) was estimated based on the content within the segmented lymph node.

Intradermal (i.d.) depot, intestine, gallbladder, and urinary bladder:
The calculation of average 18 F-sAxl intensity and total radioactivity for the six i.d.depots, as well as for the gallbladder, urinary bladder, and intestine, followed the same methodology applied to the local lymph nodes.Specifically, the average intensity for the intestine was also calculated as described above, with its total activity determined through iterative use of the isocount 3D tool.However, it is important to note the potential error due to loss of radioactivity from the gallbladder, urinary bladder, and intestine when the animals are freely moving between the PET scans.
Kidney cortex and liver: 18 F-sAxl intensity in the kidney cortex and liver was measured along two orthogonal transects and within two spherical VOIs of 1 mm and 3 mm in diameter, respectively, as illustrated in Supplementary Fig. 1.The mean intensity was calculated as described above.The total content of 18 F-sAxl (in kBq) was estimated from the mean intensity and the predicted total organ mass based on body weight, using data from Marxfeld et al 4 .The mass of the kidney cortex was assumed to be 70% of the total kidney mass.

Blood, tissue, and urine samples
Blood samples were drawn intracardially using a 0.5 ml syringe (Omnican 50, B Braun Melsungen Medical, D-34121Melsungen, Germany).The blood was then centrifuged (Eppendorf Centrifuge 5417 C, Hamburg, Germany) at 1,500 G for 10 min to obtain serum and about 40 μl was then pipetted into pre-weighed vials which were then reweighed to obtain the exact amount of serum in the vial.Tissue samples.After determination of tissue and organ weight to the nearest 0.1 mg, radioactivity was determined.Extravasation was calculated as ml plasma per g tissue by dividing (cpm per g tissue) by (cpm per ml serum).The liver and each kidney were taken out in total which allowed determination of their amount of the injected dose.Urine samples were obtained, when possible, at the end of the experiment by suprapubic puncture.Tissue and serum samples were weighed on a DeltaRange (Mettler Toledo, Spain) to the nearest 0.1 mg.The syringe for injection and blood withdrawal was a 0.5 ml syringe (Omnican 50, B Braun Melsungen Medical, D-34121 Melsungen, Germany).

Multi-angle light scattering
Multi-angle light scattering coupled to SEC (SEC-MALS) was carried out for sAXL-Fc at the core facility for Biophysics, Structural Biology, and Screening (BiSS) at the University of Bergen.Chromatography was performed using Äkta Purifier (GE Healthcare) and a Superdex 200 Increase 10/300GL column (GE Healthcare) in a running buffer containing 10 mM histidine, 150 mM NaCl, pH 6.A sample of 250 μg of sAXL-Fc was injected into the column at an isocratic flow of 0.4 mg/ml and light scattering recorded using Wyatt miniDAWN TREOS detector.The UV Absorbance recorded at 280 nm was used as a concentration source using the extinction coefficient of sAXL-Fc (0.1% Abs 1.3, calculated in ProtParam using the protein sequence).Conjugate analysis was performed in the ASTRA software (Wyatt) to obtain the molecular weight of sAXL-Fc and glycans.

SAXS Data Acquisition and Data analysis
Aliquots of sAXL-Fc (3.6 mg/ml) in phosphate buffered saline (PBS) containing 100 mM arginine were kept at -80 °C until use.Monomeric sAXL was prepared in small scale as follows.Immobilized papain (ThermoFisher) was equilibrated in digestion buffer (PBS, 20 mM cysteine, 0.5 M EDTA, pH 7) following manufacturer's instructions.sAXL-Fc was mixed with the equilibrated papain (5 mg/ml papain slurry), and the digestion mix was incubated shaking at 4 °C overnight.The reaction was stopped by centrifugation and filtration to remove immobilized papain.sAXL was separated from Fc fragments and intact sAXL-Fc by loading the digestion mix into a gravity flow column with 1 ml MabSelect Xtra (GE Healthcare) resin pre-equilibrated with PBS, pH 7. The resin was washed with 5 column volumes of PBS, and sAXL was collected in the flowthrough and initial wash fractions.The Fc fragments and intact sAXL-Fc were eluted with 50 mM sodium acetate, pH 3. SEC-SAXS data for sAXL-Fc (3.6 mg/ml), human sAXL (0.4 mg/ml), mouse sAxl (0.6 mg/ml) and monomeric BSA (1.9 mg/ml) were collected on the SWING beamline at Synchrotron Soleil (Paris, France) 5 .During the measurement, the sample was run through an Agilent Advance BioSEC 300Å column in a buffer containing 10 mM histidine, 150 mM NaCl, pH 6 (for sAXL-Fc) or 20 mM Tris, 150 mM NaCl, pH 7 (for sAXL, sAxl and BSA) at 4 °C.Initial processing was performed on the beamline using FOXTROT.The sAxl and BSA data were collected on the same day, using the same column, for best comparison.
Data were further processed in CHROMIXS 6 .All SAXS data were analyzed using the ATSAS software package 7 .Specifically, PRIMUS 8 was used for data analysis, GNOM 9 for distance distribution, and GASBOR 10 and CORAL 11 for 3D modeling.
Dummy atom models were built using DAMMIN 12 and dummy residue models with GASBOR.Fitting of the sAXL AlphaFold2 model to the SAXS data was done using CRYSOL 13 .